In proteomics, there are many methodologies using, as an additional site for a probe, a thiol group of a cysteine residue of a protein or peptide. A probe whose additional site is a thiol group has also been commercialized as, for example, a labeling kit using biotin, a fluorescent indicator, alkaline phosphatase, or the like. Such a probe can be utilized also in biochemical assays (e.g., western blotting, ELISA, intracellular fluorescent labeling) or HPLC of proteins or peptides.
Further, in proteomics, there is also a methodology in which a protein or peptide is derivatized using a probe that can label a specific amino acid residue to analyze the protein or peptide with high sensitivity. Applying to the technique of mass spectrometry, probe addition to a specific amino acid residue, is a methodology essential for enhancing ionization in mass spectrometry and reliably improving analytical accuracy.
For example, an N-terminal amino group or an amino group of a lysine residue is utilized as an additional site for a probe. There is a methodology in which, as such a probe, for example, TMPP reagent (Anal. Biochem. 2008, 380(2), 291-296 (Non-Patent Document 1)), SPITC reagent (RCM. 2004, 18(1), 96-102 (Non-Patent Document 2)), or the like is used to make it possible to select the MS/MS ion series of a peptide.
Further, a revolutionary method for quantitative analysis has been developed in which labeling using a stable isotope reagent, ICAT (Isotope-Coated Affinity Tag) is performed when an alkyl group is introduced into a thiol group of a protein or peptide (Anal. Biochem. 2001, 297, 25-31 (Non-Patent Document 3)), and the reagent has also been modified as cleavable ICAT (Mol Cell Proteomics. 2003, 2, 1198-1204 (Non-Patent Document 4)).
Further, a method for simultaneous quantitation of proteins has also been modified as iTRAQ® (Isobaric tag for relative and absolute quantitation) (Mol Cell Proteomics. 2004, 3, 1154-1169 (Non-Patent Document 5)) to analyze changes in protein or peptide expression by mass spectrometry.
In addition to the above, methods for derivatizing proteins or peptides with various probes in mass spectrometry have been reported (Anal. Chem. 1998, 70, 1544-1554 (Non-patent Document 6), Rapid Commun. Mass Spectrom. 2009; 23: 1483-1492 (Non-Patent Document 7), J. Anal. At. Spectrom., 2008, 23, 1063-1067 (Non-Patent Document 8), Anal. Chem. 1997, 69, 1315-1319 (Non-Patent Document 9), and Anal. Chem. 2004, 76, 728-735 (Non-Patent Document 10)).
On the other hand, in proteomics, a reductive alkylation method is conventionally performed after denaturation of a protein as pretreatment for effective digestion of the protein so that reoxidation of cysteine residues is prevented. Specifically, a protein that has been subjected to, for example, electrophoretic separation or denaturation with a denaturing urea solution is reduced with dithiothreitol to generate thiol groups of cysteine residues. Then, the thiol groups are alkylated with iodoacetamide, iodoacetic acid, vinylpyridine, acrylamide, or the like to block reoxidation of the thiol groups. It is understood that derivatization of cysteine residues by such a method makes it easy to unfold the chain of a protein, and as a result, enzymatic digestion as a next step easily occurs and the efficiency of the digestion is increased.